This invention relates to compositions and methods useful to identify agents that modulate the response of granulocytes to inflammatory and infectious conditions.
Granulocytes (i.e., neutrophils, eosinophils and basophils) are involved in the immune response elicited by inflammation and infection.
Inflammation
Inflammation is a localized protective response elicited by injury or destruction of tissues which serves to destroy, dilute or wall off both the injurious agent and the injured tissue. It is characterized by fenestration of the microvasculature, leakages of the elements of blood into the interstitial spaces, and migration of leukocytes into the inflamed tissue. On a macroscopic level, this is usually accompanied by the familiar clinical signs of erythema, edema, tenderness (hyperalgesia), and pain. During this complex response, chemical mediators such as histamine, 5-hydroxytryptamine, various chemotactic factors, bradykinin, leukotrienes, and prostaglandins are released locally. Phagocytic cells migrate into the area, and cellular lysosomal membranes may be ruptured, releasing lytic enzymes. All of these events may contribute to the inflammatory response.
Inflammation is initiated by, among other things, trauma, tissue necrosis, infection or immune reactions. The immediate response is temporary vasoconstriction. Vasoconstriction is followed within seconds by the acute vascular response resulting in increased blood flow (hyperemia) and edema. The acute phase is also characterized by the margination of polymorphonuclear white blood cells (neutrophils) next to endothelial cells, followed by emigration of neutrophils into the adjacent tissue. Margination is recognized by the lining up of neutrophils along the endothelium of vessels. Emigration occurs by passage of the inflammatory cells between endothelial cells.
Neutrophils
Neutrophils are the first wave of cellular attack on invading organisms and are the characteristic cells of acute inflammation. The appearance of neutrophils in areas of inflammation may be caused by chemicals released from bacteria, factors produced nonspecifically from necrotic tissue or antibody reacting with antigen. Neutrophils use an actin-rich cytoskeleton to move in a directed manner along a chemotactic gradient from the bloodstream to an inflammatory site where they ingest particles (e.g,. bacteria) and immune complexes bearing IgG (via FcR) and/or breakdown products of the complement component C3.
Neutrophils belong to a category of white blood cells known as polymorphonuclear white blood cells. The blood cells with single nuclei (mononuclear cells) form the white blood cell population that includes macrophages, T and B cells. White blood cells that contain segmented nuclei are broadly classified as polymorphonuclear. Polymorphonuclear white blood cells (or xe2x80x9cgranulocytesxe2x80x9d) are further subdivided into three major populations on the basis of the staining properties of their cytoplasmic granules in standard hematologic smears or tissue preparations: neutrophils staining pink, eosinophils staining red and basophils staining blue.
Neutrophils (also referred to as polymorphonuclear neutrophils-PMNs) make up 50% to 70% of the white blood cells (WBCs) of the peripheral blood and may be found scattered diffusely in many tissues, although they are most frequently found in areas of acute inflammation or acute necrosis. Like other WBCs, neutrophils are produced from precursor cells in the bone marrow and released into the blood when mature. After entering the circulation, neutrophils are thought to last only 1 or 2 days.
Neutrophils are characterized by numerous cytoplasmic granules that contain highly destructive enzymes that must be kept isolated from the cytoplasm. These granules contain a number of oxygen-independent enzymes as well as oxygen-dependent mechanisms of killing. Upon attraction to sites of inflammation, neutrophils attempt to engulf and digest bacteria coated with antibody and complement. Phagocytosis by neutrophils is also usually accompanied by release of the lysosomal enzymes into the tissue spaces, particularly if the organism is difficult for the neutrophil to digest
At least three cytoplasmic granules are identifiable in neutrophils: specific granules containing lactoferrin, B cytochrome, the complement receptor CR3 and xcexc2-integrin; azurophilic granules containing acid hydrolases and other enzymes; and a third granule containing gelatinase.
In addition to the role neutrophils and other granulocytic cells play in immune response to pathogens, including bacterial infection, neutrophils and other granulocytic cells play an unwanted role in many chronic inflammatory diseases. There are many disease states in which excessive or unregulated granulocytic cell infiltration and activation are implicated in exacerbating and/or causing the disease. For instance, many inflammatory diseases are characterized by massive neutrophil infiltration, such as psoriasis, inflammatory bowel disease, Crohn""s disease, asthma, cardiac and renal reperfusion injury, adult respiratory distress syndrome, rheumatoid arthritis, thrombosis and glomerulonephritis. All of these diseases are associated with increased IL-8 production which may be responsible for the chemotaxis of neutrophils into the inflammatory site.
While the role of neutrophil infiltration and activation in inflammation is well known, the biosynthetic responses of neutrophils to pathogens, chemotactic agents, proinflammatory molecules, etc. are not as well understood. Neutrophils were once thought to be in a state of terminal differentiation, thereby lacking biosynthetic ability. This view is consistent with the relative scarcity in mature circulating neutrophils of ribosomes and endoplasmic reticulum and with the ability of neutrophils to ingest particles when RNA and/or protein synthesis has been inhibited. More recently it has been demonstrated that neutrophils perform more active roles in their response to environmental stimuli.
It has thus recently been established that neutrophils synthesize de novo important macromolecules including, but not limited to interleukin (IL) 1, I1-6, I1-8, tumor necrosis factor (TNFxcex1), granulocyte and macrophage colony-stimulating factors, interferon xcex1 (IFNxcex1), intercellular adhesion molecule (ICAM-1) and membrane and cystoskeletal molecules, such as major histocompatibility class I antigens and actin (Beaulieu et al (1992) J. Biolog. Chem. 267(1):426-432; Arnold et al. (1993) Infect. Immun. 61(6):2545-2552; and Elsner et al. (1995) Immunobiol 193:456-464). No study, however, has taken a systematic approach to assess the transcriptional response during neutrophil activation via contact with a pathogen or from neutrophils isolated from a subject with a sterile inflammatory disease.
Eosinophils and Basophils
Eosinophils are another granulocytic or polymorphonuclear white blood cell that are involved in the inflammatory response. Eosinophils are found predominately in two types of inflammation: allergy and parasite infections.
The role of eosinophils in the host response to parasites is thought to be mediated through the components of the eosinophilic granules. Eosinophils are cytotoxic to schistosome larvae through an antibody-dependent cell-mediated mechanism. Eosinophil cationic proteins are highly toxic for schistosomes and may be responsible for binding of eosinophils to parasitic worms as well as fragmentation of the parasite.
The role of eosinophils in acute inflammation is not fully understood. On one hand, there is evidence that enzymes in eosinophils may serve to limit the extent of inflammation by neutralizing mediators of anaphylaxis, such as LTC4, histamine and platelet-activating factor. On the other hand, there is increasing evidence that cationic proteins in eosinophilic granules are mediators of acute inflammation. Eosinophil activation is associated with acute tissue injury and cause an intense vasoconstriction in lung microvasculature, followed by increased pulmonary vascular permeability and pulmonary edema.
Basophils or mast cells are the other major cell type characterized as a granulocytic or polymorphonuclear white blood cell. Mast cells contain granules with a variety of biologically active agents which, when released extracellularly (degranulation), cause dilation of the smooth muscle of arterioles (vasodilation), increased blood flow, and contraction of endothelial cells, thereby opening up vessel walls to permit egress of antibodies, complement or inflammatory cells into tissue spaces.
While the role of neutrophils and other granulocytic cells in inflammation and/or the immunological response to infection has been the subject of intense study, little is known about the global transcriptional response of granulocytes during cell activation. The present inventors have devised an approach to systematically assess the transcriptional response from granulocytic cells activated through contact with a pathogen or from granulocytic cells isolated from a subject with a sterile inflammatory disease.
The present invention includes a method to identify granulocytic cell genes that are differentially expressed upon exposure to a pathogen by preparing a gene expression profile of a granulocytic cell population exposed to a pathogen and comparing that profile to a profile prepared from quiescent granulocytic cells. cDNA species, and therefore genes, which are expressed de novo upon neutrophil contact with a pathogen are thereby identified. The present invention is particularly useful for identifying cytokine genes, genes encoding cell surface receptors and genes encoding intermediary signaling molecules.
The present invention also includes a method to identify granulocytic cell genes that are differentially expressed in response to a sterile inflammatory disease by preparing a gene expression profile of a granulocytic cell population isolated from a subject exhibiting the symptoms of a sterile inflammatory disease and comparing that profile to a profile prepared from granulocytic cells isolated from a normal granulocytic cell population. cDNA species, and therefore genes, which are differentially expressed in the granulocytic cells of a subject exhibiting the symptoms of a sterile inflammatory disease are thereby identified.
The present invention also includes a method to identify granulocytic cell genes that are differentially expressed upon exposure of a granulocytic cell population to an agonist (pro-inflammatory molecule) by preparing a gene expression profile of a granulocytic cell population contacted with an agonist and comparing that profile to a profile prepared from noncontacted granulocytic cells, thereby identifying cDNA species, and therefore genes, which are expressed de novo in the granulocytic cells contacted with the agonist are thereby identified.
The present invention further includes a method to identify a therapeutic or prophylactic agent that modulates the response of a granulocyte population to a pathogen, comprising the steps of preparing a first gene expression profile of a quiescent granulocyte population, preparing a second gene expression profile of a granulocyte population exposed to a pathogen, treating said exposed granulocyte population with the agent, preparing a third gene expression profile of the treated granulocyte population, comparing the first, second and third gene expression profiles and identifying agents that modulate the response of a granulocyte population to the pathogen.
Another aspect of the invention is a method to identify a therapeutic agent that modulates the expression of genes in a granulocyte population found in a subject having Another aspect of the invention includes a method to identify a therapeutic or prophylactic agent that modulates the response of a granulocyte cell population in a subject having a sterile inflammatory disease, comprising the steps of preparing a first gene expression profile of a granulocyte population in a subject having a sterile inflammatory disease, treating the granulocyte population with the agent, preparing a second gene expression profile of the treated granulocyte population, comparing the first and second gene expression profiles with the gene expression profile of a normal granulocyte population and identifying agents that modulate the expression of genes whose transcription levels are altered in the granulocyte population of the subject as compared with normal granulocyte population.
A further aspect of the present invention is a method to identify a therapeutic or prophylactic agent that modulates the response of a granulocytic population to an agonist (pro-inflammatory molecule), comprising the steps of preparing a first gene expression profile of a quiescent granulocyte population, preparing a second gene expression profile of a granulocyte population exposed to an agonist, treating the exposed granulocyte population with the agent, preparing a third gene expression profile of the treated granulocyte population, comparing the first, second and third gene expression profiles and identifying agents that modulate the response of a granulocytic population exposed to an agonist.
The present invention also includes a method of diagnosing the exposure of a subject to a pathogen, comprising the steps of preparing a first gene expression profile of a granulocyte population from the subject, comparing the first gene expression profile to a second gene expression profile of a granulocyte population exposed to that pathogen and to a third gene expression profile of a normal granulocyte preparation and diagnosing whether the subject has been exposed to a pathogen.
Another aspect of the invention includes a method of diagnosing a sterile inflammatory disease in a subject, comprising the steps of preparing a first gene expression profile of a granulocyte population from the subject, comparing the first gene expression profile to at least one second gene expression profile from a granulocyte population from a subject having a sterile inflammatory disease and to a third gene expression profile of a normal granulocyte preparation and thereby determining if the subject has a sterile inflammatory disease.
The present invention also includes a method of identifying new bacterial virulence factor genes by preparing a first gene expression profile of a quiescent granulocyte population, preparing a second gene expression profile of a granulocyte population exposed to a virulent or avirulent bacterial strain, preparing a third gene expression profile from a granulocyte population exposed to a bacterial strain with a mutation in a putative bacterial virulence factor gene, comparing the first, second and third gene expression profiles and identifying a bacterial virulence factor gene.
Another aspect of the invention is a composition comprising a grouping of nucleic acids that correspond to at least a part of one or more of the genes whose expression levels are modulated in a granulocyte population that has been exposed to a pathogen, these nucleic acids being affixed to a solid support.
Lastly, an aspect of the invention is a composition comprising a grouping of nucleic acids that correspond to at least part of one or more genes whose expression levels are modulated in a granulocyte population found in a subject having a sterile inflammatory disease, these nucleic acids being affixed to a solid support.